Although, with few exceptions, the genetic codes of all known organisms encode the same twenty amino acids, all that is required to add a new amino acid to the repertoire of an organism is a unique tRNA/aminoacyl-tRNA synthetase pair, a source of the amino acid, and a unique selector codon that specifies the amino acid (Furter (1998) Protein Sci., 7:419-426). Previously, we have shown that the amber nonsense codon, TAG, together with orthogonal M. jannaschii and E. coli tRNA/synthetase pairs can be used to genetically encode a variety of amino acids with novel properties in E. coli (Wang et al., (2000) J. Am. Chem. Soc., 122:5010-5011; Wang et al., (2001) Science, 292:498-500; Wang et al., (2003) Proc. Natl. Acad. Sci. U.S.A., 100:56-61; Chin et al., (2002) Proc. Natl. Acad. Sci. U.S.A., 99:11020-11024), and yeast (Chin and Schultz, (2002) ChemBioChem, 3:1135-1137), respectively. The limited number of noncoding triplet codons, however, severely restricts the ultimate number of amino acids encoded by any organism.
There are many examples of naturally occurring +1 frameshift suppressors including UAGN suppressors derived from Su7 encoding glutamine (Magliery et al., (2001) J. Mol. Biol., 307:755-769), sufJ-derived suppressors of ACCN codons encoding threonine (Anderson et al., (2002) Chem. Biol., 9:237-244) and CAAA suppressors derived from tRNALys and tRNAGln (Anderson and Schultz, (2003) Biochemistry, 42(32):9598-608). Moreover, genetic selections have been used to identify efficient four- and five-base codon suppressor tRNAs from large libraries of mutant tRNAs, including an E. coli tRNAUCCUSer suppressor (Ibba et al., (1999) Proc. Natl. Acad. Sci. U.S.A., 96:418-423; Kwok and Wong, (1980) Can. J. Biochem., 58:213-218). This natural phenomena has been extended for unnatural amino acid mutagenesis in vitro using chemically aminoacylated tRNAs. A variety of amino acids, including fluorophore/quencher pairs (Terada et al., (2002) Nat. Struct. Biol., 9:257-262), have been incorporated into protein in response to AGGU and CGGG (Hou et al., (1992) Biochemistry, 31:4157-4160; Yarus et al., (1986) J. Mol. Biol., 192:235-255; Miller, (1972) Experiments in molecular genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). To further expand the genetic code, there is a need to develop improved and/or additional components of the biosynthetic machinery, e.g., orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases and/or unique codons. This invention fulfills these and other needs, as will be apparent upon review of the following disclosure.